Biochemistry and Cell Biology

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Browsing Biochemistry and Cell Biology by Author "Agbandje-McKenna, Mavis"

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    Random Insertion of mCherry Into VP3 Domain of Adeno- associated Virus Yields Fluorescent Capsids With no Loss of Infectivity
    (American Society of Gene & Cell Therapy, 2012) Judd, Justin; Wei, Fang; Nguyen, Peter Q.; Tartaglia, Lawrence J.; Agbandje-McKenna, Mavis; Silberg, Jonathan J.; Suh, Junghae
    Adeno-associated virus (AAV)-derived vectors are promising gene delivery systems, and a number of design strategies have been pursued to improve their performance. For example, genetic insertion of proteins into the capsid may be used to achieve vector retargeting, reduced immunogenicity, or to track vector transport. Unfortunately, rational approaches to genetic insertion have experienced limited success due to the unpredictable context-dependent nature of protein folding and the complexity of the capsid's macroassembly. We report the construction and use of a frame-enriched DNase-based random insertion library based on AAV2 cap, called pAAV2_RaPID (Random Peptide Insertion by DNase). The fluorescent mCherry protein was inserted randomly throughout the AAV2 capsid and the library was selected for fluorescent and infectious variants. A capsid site was identified in VP3 that can tolerate the large protein insertion. In contrast to previous efforts to incorporate fluorescent proteins into the AAV2 capsid, the isolated mCherry mutant maintains native infectivity while displaying robust fluorescence. Collectively, these results demonstrate that the pAAV2_RaPID platform library can be used to create fully infectious AAV vectors carrying large functional protein domains on the capsid.
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    Tunable Protease-Activatable Virus Nanonodes
    (American Chemical Society, 2014) Judd, Justin; Ho, Michelle L.; Tiwari, Abhinav; Gomez, Eric J.; Dempsey, Christopher; Vliet, Kim Van; Igoshin, Oleg A.; Silberg, Jonathan J.; Agbandje-McKenna, Mavis; Suh, Junghae
    We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virusﾖreceptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery.